Methods and devices for sample analysis

ABSTRACT

Methods and devices for cytometric analysis are provided. A cytometry apparatus is provided which may be used with a stationary sample cuvette for analysis of a stationary sample or with a flow sample cuvette for analysis of a flowing sample. The methods and devices provided herein may be used to perform cytometric analysis of samples under a wide range of experimental and environmental conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This applications claims the benefit of, and priority to U.S.Provisional Patent Application No. 61/837,167, filed Jun. 19, 2013, thecontent of which is hereby incorporated by reference in its entirety forall purposes.

BACKGROUND

Cytometric analysis of a sample can provide a wealth of informationabout the sample, such as the number and type of cells or otherparticles in the sample, specific molecules which may be present in thesample, and other information.

While a large number of systems and devices for the analysis of samplesare known, there is a need for improved systems for cytometric analysis.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY

Provided herein are devices and methods for sample analysis.

In an embodiment, a cytometry apparatus is provided. The cytometryapparatus may be configured to receive cytometry cuvettes which areconfigured for analysis of stationary or flowing samples. The cytometryapparatus may be used to obtain information from stationary or flowingsamples.

In embodiments, the cytometry apparatus may direct light from a sampleto a first optical pathway or a second optical pathway, where the firstoptical pathway contains one or more image sensors and the secondoptical pathway contains one or more light detectors.

In embodiments, devices and methods provide herein may be used andperformed in micro-gravity environments.

In embodiments, provided herein is a cytometry apparatus comprising: astage, wherein the stage is configured to receive and support acytometry cuvette, wherein the cytometry cuvette is configured toreceive and support a biological sample, and wherein the cytometrycuvette comprises a sample plane on which the biological sample issupported; an objective, wherein the objective comprises an entrancepupil and an exit pupil, and wherein the objective is optically coupledto the stage such that light from the biological sample supported by thecytometry cuvette supported by the stage may enter the entrance pupiland exit the exit pupil; a first detection train, wherein the firstdetection train comprises one or more image sensors; a second detectiontrain, wherein the second optical pathway comprises one or more lightdetectors; an actuatable structure for directing light, wherein theactuatable structure is configured to direct light from the exitaperture of the objective to the first detection train or the seconddetection train; and an illumination train, wherein the illuminationtrain comprises a light source, wherein the illumination train isconfigured to provide at least a first illumination output and a secondillumination output, and wherein for the first illumination output theillumination train is configured to focus light of a first range ofwavelengths from the light source on a first location on the sampleplane of the cytometry cuvette and the illumination train is configuredto focus light of a second range of wavelengths from the light source ona second location on the sample plane of the cytometry cuvette. Otherrelated cytometry apparatuses, systems and methods are also provided.

In embodiments, in systems and methods provided herein, a sample may beloaded into a cytometry cuvette before the cuvette is moved to a stageof a cytometry apparatus, or after the cuvette is moved to the stage ofthe cytometry apparatus.

In embodiments, a stage of a cytometry apparatus provided herein mayhave an opening. In embodiments, the opening may have no material in theopening. In other embodiments, the opening may have an opticallytransmissive material (e.g. glass, clear plastic, etc.).

In embodiments, a sample provided herein may contain multiple cells.Multiple cells typically includes at least two cells. In embodiments,context indicate that a sample described herein as containing “multiplecells” may contain more than two cells, such as at least 4, 5, 6, 7, 9,10, or more cells.

In embodiments a focal point in a flow cytometry cuvette has a diameterof at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200microns, a diameter of no more than 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, or 300 microns, or a diameter of at least 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, or 200 microns and no more than 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 microns. In embodimentsthe space between focal points in a flow channel may be least 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 microns, no more than 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 microns, or atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 microns andno more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300microns.

In embodiments, a light source provided herein may provide continuouslight, or it may intermittently provide light. For example, it mayprovide flashes of light to coincide with the timing of the movement ofcells through a flow channel of a flow sample cuvette.

In embodiments, references herein to “cells” in systems and methodsprovided herein also apply to similarly sized and shaped small objects(e.g. particles, beads, etc.), unless the context dictates otherwise.

Other goals and advantages of the invention will be further appreciatedand understood when considered in conjunction with the followingdescription and accompanying drawings. While the following descriptionmay contain specific details describing particular embodiments of theinvention, this should not be construed as limitations to the scope ofthe invention but rather as an exemplification of preferableembodiments. For each aspect of the invention, many variations arepossible as suggested herein that are known to those of ordinary skillin the art. A variety of changes and modifications can be made withinthe scope of the invention without departing from the spirit thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

FIG. 1 shows an exemplary schematic of a cytometry apparatus.

FIG. 2 shows an exemplary schematic of a stationary sample cuvette.

FIGS. 3A and 3B show an exemplary schematic of a flow sample cuvette; 3Ashows a side view and 3B shows a top-down view.

FIG. 4 shows an exemplary schematic of an image that can be obtained byan image sensor of a cytometry apparatus.

It is noted that the drawings and elements therein are not necessarilydrawn to shape or scale. For example, the shape or scale of elements ofthe drawings may be simplified or modified for ease or clarity ofpresentation. It should further be understood that the drawings andelements therein are for exemplary illustrative purposes only, and notbe construed as limiting in any way.

DETAILED DESCRIPTION

While various embodiments of the invention are shown and describedherein, these are provided by way of example only. It should beunderstood that there is no intent to limit the invention to theparticular forms disclosed, but rather, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention provided herein.

As used herein, references to a “sample” refer to the sample as a wholeor any portion thereof, unless the context clearly dictates otherwise.For example, a description of an image of a “sample” may refer to animage of a single cell within the sample, or it may refer to an image ofa field of view of a sample within a cuvette, which may contain, forexample, one or more cells, no cells, one or more non-cellularparticles, or any combination thereof.

As used herein, descriptions of objects being in “optical communication”refer to objects which are spatially positioned such that they may bedirectly or indirectly optically coupled. An object which is in opticalcommunication with another object may, for example, emit light, reflectlight, transmit light, or absorb light. Objects which are in opticalcommunication may be positioned from each other in a straight path (e.g.a light source and a light sensor may be in a straight line relative toeach other, such that light travels in a straight line between the lightsource and light sensor), or they may be at an angle to each other, withone or more structures positioned between the objects which to changethe direction of movement of light (e.g. a light source and a lightsensor may be at a 90 degree angle relative to each other, with anangled mirror being situated in the light path between the light sourceand light sensor, such that light from the light source is reflected offthe mirror at a 90 degree angle towards the light sensor). Also, lightdoes not need to be continuously moving between two objects for them tobe considered to be in optical communication. For example, a lightsource and light sensor are considered to be in optical communication ifthe light source and light sensor are situated such that light from thelight source can reach the light sensor under selected conditions; lightfrom the light source does not need to continuously reach the lightsensor for them to be considered to be in optical communication. Inanother example, an object which may reflect light (e.g. a cytometrycuvette) may be considered to be in optical communication with a lightsource which emits light which strikes the object or with a light sensorwhich absorbs light reflected by the object. As used herein, objectsdescribed as being in “optical communication” may also be described asbeing “optically coupled”.

In embodiments, provided herein is a cytometry apparatus. The cytometryapparatus may be configured to obtain at least a 2-dimensional or3-dimensional image of a sample. The cytometry apparatus may beconfigured to obtain multiple images of a sample over a period of time.The cytometry apparatus may alternatively or additionally be configuredto detect light of one or more selected wavelength(s) emitted by orscattered by a sample. A cytometry apparatus may be used to obtaininformation relating to, for example, cells, crystals, particles, orother small objects.

The cytometry apparatus may contain a stage. The stage may be configuredto receive a cytometry cuvette, slide, or other structure which maycontain a sample for cytometric analysis. The stage may contain anopening through which light may pass between a cuvette and an objective(discussed further below) adjacent to or in optical communication withthe cuvette.

The cytometry apparatus may contain one or more light sources. The lightsource may be, for example, a tungsten bulb, a tungsten-halogen bulb, anarc lamp (e.g. mercury, xenon, zirconium, or metal halide), a laser(e.g. argon-ion laser, krypton-ion laser), or a light-emitting diode. Alight source may emit light which has approximately uniform brightnessacross the wavelengths of the visible spectrum (e.g. white light), or itmay emit light of variable brightness across the visible spectrum (e.g.a lamp, such as a mercury arc lamp, may emit light having brightnesspeaks at 365, 405, 436, 546, and 579 nm). In embodiments, a light sourcemay emit light having a single peak of brightness in the visiblespectrum. For example, LEDs may emit light of only a single brightnesspeak, which may have a spectral width (i.e. the width of the emissionpeak at 50% maximum intensity/full width at half maximum (FWHM)) of, forexample, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, or 200 nm. Different LEDs may have brightness peaks at differentwavelengths. For example, an LED may have brightness peak at, forexample, 365, 400, 445, 455, 465, 470, 505, 525, 530, 535, 565, 585,590, 595, 625, or 635 nm.

In embodiments, the cytometry apparatus may contain 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 50, 100, 500, 1000, ormore light sources. In a cytometry apparatus containing two or morelight sources, the light sources may be all of the same type (e.g. alllasers or all LEDs), or the light sources may be of two or moredifferent types (e.g. a tungsten bulb and a mercury arc lamp). In acytometry apparatus containing two or more light sources, the lightsources may have the same or different emission brightness peak(s).

The cytometry apparatus may contain an objective. The objective maycontain one or more lenses and may serve to capture light from a samplein a cuvette on the stage. The objective may also be positioned in theapparatus such that light from the light source(s) is directed into andthrough the objective to a cuvette on the stage. In embodiments, theobjective may also function as a condenser such that the light isfocused onto the sample in the cuvette as it passes through theobjective. The position of the objective, the stage, the cuvette, orother component of the cytometry apparatus may be adjusted in order tofocus the light from the objective onto the sample or to collect lightfrom the sample.

In embodiments, a light source may be in-line with the objective, suchthat light travels in a straight path from the light source to theobjective. In other embodiments, a light source may be at an angle tothe objective, such light is directed in a non-straight path from thelight source to the objective. For example, a light source may besituated at a 90 degree angle from the aperture of the objective, andthe light from the light source may be directed to the aperture of theobjective by, for example, a mirror or fiber optics. In embodiments, amirror which directs light from a light source into the objective may bea dichroic mirror. A dichroic mirror may reflect light above or below agiven cutoff wavelength, and may transmit light on the opposite side ofthe cutoff wavelength. For example, a particular dichroic mirror mayhave a cutoff wavelength of 425 nm, and it may transmit 90% of light at440-700 nm and reflect 90% of light at 380-410 nm. In another example,another dichroic mirror may also have a cutoff wavelength of 425 nm, butit may reflect 90% of light at 440-700 nm and transmit 90% of light at380-410 nm. Dichroic mirrors are available, for example, from Thorlabs,Inc. (Newton N.J.). As used herein, “dichroic mirrors” also may includemirrors that are polychroic—i.e. which transmit or reflect multipleranges of wavelengths of light.

The objective may also be configured to capture light emitted from orscattered by the sample. For example, light of a first wavelength may bedirected through the objective onto a sample in a cuvette on the stage.The sample may contain a fluorescent molecule which is excited by lightof the first wavelength. Upon excitation by light of the firstwavelength, the fluorescent molecule may emit light of a secondwavelength. The emitted light from the fluorescent molecule may travelback through the objective and into the interior of the cytometryapparatus.

In embodiments, light passing from the sample through the objective mayencounter a dichroic mirror downstream from the objective (i.e. afterpassing through the objective). The dichroic mirror may be the samemirror which reflected light from the light source into the aperture ofthe objective. Light of one or more wavelengths from the objective maybe transmitted through the dichroic mirror. As discussed above,different dichroic mirrors may transmit or reflect different wavelengthsor ranges of wavelengths of light.

The cytometry apparatus may contain one or more sensors for detectinglight emitted, reflected, or scattered from a sample in a cuvette.Sensors for detecting light (also referred to herein as “opticalsensors”) may include image sensors and light detectors.

In embodiments, the cytometry apparatus may contain an image sensorcapable of obtaining at least a two-dimensional image of an object. Asused herein, an “image sensor” refers to a device which can convert anoptical image into one or more electrical signals. Image sensorsinclude, for example, charge-coupled device (CCD), complementarymetal-oxide semiconductor (CMOS), and hybrid CCD/CMOS (e.g. sCMOS)sensors. Digital cameras contain an image sensor such as a CCD or CMOSsensor, and, in embodiments, a digital camera itself may be referred toas an image sensor. Generally, a single image sensor contains an arrayof a large number of photoactive structures (e.g. capacitors,photodiodes), such that when the image sensor is exposed to light, eachof the many photoactive structures generates an electric chargeproportional to the light intensity on the respective structure, therebyinitiating the conversion of an image into an electrical signal. Inembodiments, the photoactive structures of an image sensor may bereferred to as “pixels”, and an image sensor may be referred to as a“pixelated” sensor. In embodiments, an image sensor may be used togenerate a three-dimensional image of an object, by, for example, usinga processor to combine multiple optical images obtained with an imagesensor. In embodiments, an image sensor may be used to generate an imageof an object over time, by collecting multiple images of the object withthe image sensor. An image sensor may be used to record video of asample.

In embodiments, the cytometry apparatus may contain a light detector. Asused herein, a “light detector” refers to a non-pixelated/non-arrayedlight sensor. An individual “light detector” as used herein is capableof detecting photons of light, but not of converting an optical imageinto an electrical signal. Examples of “light detectors” as used hereininclude, for example, photomultiplier tubes (PMT), photodiodes, andavalanche photodiodes. In embodiments, a light detector may have greaterlight sensitivity than an image sensor. In embodiments, a light detectormay be optically coupled to one or more optical filters such that onlylight a selected range of wavelengths is permitted to reach the lightdetector.

In embodiments, light which has been emitted or scattered from a sampleand passed through the objective of a cytometry apparatus providedherein may be directed into an optical pathway in which the light may beselectively directed into a first optical pathway towards one or moreimaging sensors or into a second optical pathway towards one or morelight detectors. Light may be selectively directed into a first orsecond optical pathway by use of one or more structure for the directionof light such as, for example, a mirror, fiber optics, prism, lens,filter, or a combination thereof. In embodiments, a structure fordirecting the path of light may be coupled to an actuation mechanism(e.g. an electric motor, a pneumatic actuator, hydraulic piston, relay,comb drive, piezoelectric actuator, thermal bimorph, digitalmicromirror, or an electroactive polymer). The actuation mechanism maybe coupled to a controller. The controller may be configured to receiveprotocols or instructions from, for example, a user, local memory, or anexternal database. The controller may provide instructions to theactuation mechanism to change the position of the hardware for directingthe path of light, in order to control the direction of the light.

In embodiments, an optical pathway in which light may be selectivelydirected towards an imaging sensor or light detector may contain one ormore mirrors to direct light towards a selected optical sensor. A mirrorin an optical pathway may be used in various different ways to directlight towards a selected pathway or sensor. For example, in embodiments,a mirror may be movable by an actuation mechanism such that in a firstposition it is in the path of light coming from the objective of thecytometry apparatus (and it therefore reflects the light from theobjective), and in a second position it is not in the path of lightcoming from the objective of the cytometry apparatus (and it thereforedoes not reflect or otherwise interfere with the light from theobjective). By such a mechanism, a mirror may selectively directinglight from the objective to a first or a second optical pathway. Inanother example, in embodiments, a mirror may be movable by an actuationmechanism such that in a first position it reflects the light from theobjective at a first angle, and in a second position it reflects lightthe from the objective at a second angle, thereby selectively directinglight from the objective to a first or a second optical pathway.

In embodiments, an optical pathway containing one or more lightdetectors may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, ormore light detectors. A plurality of light detectors in an apparatus maybe arranged such that different detectors of the plurality are eachpositioned to receive light of different ranges of wavelengths. Forexample, an optical pathway containing three light detectors may containa first light detector configured to receive light of 400-500 nm, asecond light detector configured to receive light of 500-600 nm, and athird light detector configured to receive light of 600-700 nm.

Various materials may be used in an optical pathway containing one ormore light detectors in order to restrict the wavelength(s) of lightthat reach a given light detector. For example, a light detector may bebehind a filter or monochromator, which may transmit light of only alimited range of wavelengths (blocking the transmission of light ofwavelengths outside of the limited transmission range). In anotherexample, a dichroic mirror may be provided in an optical pathwaycontaining one or more light detectors. The dichroic mirror may bepositioned such that, for example, it reflects light of certainwavelengths to a light detector or such that it transmits light ofcertain wavelengths to a light detector. In embodiments, an opticalpathway containing a dichroic mirror may contain two or more lightdetectors, such that light reflected by the dichroic mirror is detected,and also such that light transmitted by the dichroic mirror is detected.

As used herein, an “optical pathway” may also be referred to as an“optical train”. Also, an optical train containing materials and aconfiguration specialized for emitting light may be referred to hereinas an “illumination train”, and an optical train containing materialsand a configuration specialized for detecting light may be referred toas a “detection train”. An optical train may include, for example, oneor more of and any combination of lenses, mirrors, prisms, gratingelements, light sources, light sensors, or other materials for guidingthe movement of, emitting, or detecting light. An illumination traintypically comprises at least a light source, and a detection traintypically comprises at least an optical sensor.

In embodiments, a cytometry apparatus may contain one or more imagesensors and one or more light detectors. In embodiments, the objectiveof the cytometry apparatus may be configured to be in opticalcommunication with both image sensor(s) and light detector(s), such thatlight emitted or reflected from a sample through the objective may bedetected by one or more image sensor or light detector. In someembodiments, the cytometry apparatus may be configured such that lightfrom the objective is directed to either the image sensor(s) or lightdetector(s). In other embodiments, the cytometry apparatus may beconfigured such that some light from the objective is directed to theimage sensor(s) (e.g. light of a first range of wavelengths) and otherlight from the objective is directed to the light detector(s) (e.g.light of a second range of wavelengths). The cytometry apparatus maycontain one or more mechanisms to direct light from the sample to theimage sensor(s) and/or the light detector(s), as discussed above.

The cytometry apparatus may also contain light sources and opticalsensors which are not in optical communication with the objective of theapparatus. For example, the cytometry apparatus may contain one or morelight sources or optical sensors which are in the same or similar planeas a sample in a cuvette on the stage. The plane may be parallel to, orat a different angle (e.g. 15, 30, 45, 60, 75, or 90 degrees) relativeto the cuvette. This type of light source or detector may be referred toherein as a “sample coplanar” or “non-objective-linked” light source oroptical sensor, and may have any of the features of light sources oroptical sensors described elsewhere herein. In some embodiments, asample coplanar/non-objective-linked light source or optical sensor mayhave any orientation in which it is in optical communication with asample in a cuvette. In embodiments, a cuvette for use with a cytometryapparatus may contain one or more openings or optical pathways to permitlight from a sample coplanar light source to reach the sample in thecuvette or to permit light scattered or emitted from the sample totravel to a sample coplanar optical sensor. In an embodiment, a samplecoplanar light source generates white light and a corresponding samplecoplanar optical sensor detects light across the visible spectrum. Inembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sample coplanarlight sources or optical sensors may be provided in relation to acytometry apparatus stage. In some embodiments, fiber optics may be usedto connect an optical pathway to or from a cuvette to a sample coplanaroptical sensor or light source. Through the use of fiber optics, two ormore optical pathways in a cuvette may be operably connected to a singlesample coplanar optical sensor or light source. For example, a first endof a fiber optic cable may be permanently attached to an optical sensoror light source. However, the second of the cable may be moveable suchthat it may be attached to different optical pathways in the cuvette.This may permit multiple optical configurations to be generated with thesame cuvette. In some embodiments, a sample coplanar optical sensor is alight detector, as described herein.

In embodiments, sample coplanar light sources or optical sensors may beprovided which are within a housing of the cytometry apparatus. In otherembodiments, sample coplanar light sources or optical sensors may beprovided which are not within a housing of the cytometry apparatus. Instill other embodiments, sample coplanar light sources or opticalsensors may be provided which are not part of the cytometry apparatus,but which are in electrical or optical communication with the apparatus.

A schematic of an example cytometry apparatus is provided in FIG. 1.FIG. 1 shows a side view of the exemplary cytometry apparatus. Theexemplary cytometry apparatus 1001 may have a housing 1011 and a stage1012. The stage 1012 may be configured to receive and support a cuvette1021. The stage 1012 may contain an opening 1013 through which light maytravel from an objective 1031 to a sample in a cuvette 1021 and from asample in a cuvette 1021 to an objective 1031 in the cytometryapparatus. The stage 1012 may also contain one or more supports 1014 toaid in the positioning of a cuvette on the stage. A dichroic mirror 1032may be present and situated so as to reflect to the objective 1031 lightof certain wavelengths from one or more light sources 1033, 1034, 1035and to transmit light from the sample (and the objective) of certainwavelengths. In the exemplary apparatus of FIG. 1, three light sources1033, 1034, and 1035 are present. The light sources 1033, 1034, 1035 areLEDs, each with a different range of emission wavelengths. The lightsources 1033, 1034, 1035 may be arranged in a larger compound lightsource 1039 which contains one or more of the light sources 1033, 1034,1035 and which may direct light from the one or more light sources, suchas through the use of dichroic mirrors 1036, 1037, 1038. Light from thelight source 1039 may have any configuration; for example, although FIG.1 depicts the light as being focused, with a focal point at the dichroicmirror 1032, the light from the light source may be collimated and notfocused on the mirror 1032. Also, in embodiments, collimated light maybe reflected from the mirror into the objective. In certainconfigurations, light from the objective may focus light of one or moreselected ranges of wavelengths onto one or more locations. For example,in FIG. 1, light of a first selected range of wavelengths from the lightsource is focused on a first location, light of a second selected rangeof wavelengths from the light source is focused on a second location,and light of a third selected range of wavelengths from the light sourceis focused on a third location (collectively indicated by 1080). Amirror 1040 may be included which is configured to reflect lighttransmitted through the dichroic mirror 1032. Also, although notdepicted in FIG. 1, in embodiments, one or more actuatable lens may bepresent, which may be moved between the light source 1039 and the mirror1040. Positioning of the actuatable lens between the light source 1039and the mirror 1040 may configure light for use with obtaining images ofstationary samples with an image sensor. The cytometry apparatus maycontain an actuatable mirror 1041, which can be moved to selectivelydirect light from the objective to either a first optical pathway 1042towards one or more imaging sensors or to a second optical pathway 1043towards one or more light detectors. In the exemplary apparatus of FIG.1, the actuatable mirror 1041 may be moved to at least a first positionor a second position. In the first position (not shown), the actuatablemirror 1041 is positioned such that it does not interfere with themovement of light from the mirror 1040 (i.e. it is positioned out of theway of the path of light), and, as a result, the light travels along thefirst optical pathway 1042 which leads to an image sensor 1051. In thesecond position (shown), the actuatable mirror 1041 is position suchthat it reflects light from the 1040 (i.e. it is position in the way ofthe path of the light), and, as a result, the light is directed to thesecond optical pathway 1043, which leads to a collection of lightdetectors 1052, 1053, 1054, and 1055. Accordingly, light from theobjective may be selectively directed towards the first optical pathway1042 or the second optical pathway 1043. Dichroic mirrors 1056, 1057,1058, 1059 may be included in the second optical pathway 1043. Thedichroic mirrors 1056, 1057, 1058, 1059 in the second optical pathwaymay separate the light in the second optical pathway into multipledifferent light paths, based on the light wavelength. The light ofdifferent wavelength ranges may, in turn, be directed to different lightdetectors 1052, 1053, 1054, 1055. In embodiments, the first opticalpathway 1042, second optical pathway 1043, or portions thereof mayfurther include one or more filters 1061 or lenses 1062 (for clarity,only a single filter and lens is notated in the figure). The filter 1061may transmit only a limited range of wavelength(s), and the lens 1062may focus the light onto the image sensor or light detector.

The exemplary cytometry apparatus of FIG. 1 may further contain a samplecoplanar light source 1071 and a sample coplanar optical sensor 1072.

The stage of the cytometry apparatus may be configured to receive andsupport a variety of different structures which may contain a sample forcytometric analysis. Structures which may contain a sample may bereferred to, for example, as cytometry cuvettes or slides. Typically, astage will support a cytometry cuvette by the cuvette resting on top ofthe stage. In other embodiments, a stage may be configured support acytometry cuvette from above the cuvette, such as by containing a slotfor the cuvette, or by having structures to attach a cuvette (e.g.magnets, screws, clamps, etc.). A cytometry cuvette or slide whichcontains a sample may also be described as “supporting” a sample.Samples may be loaded into cytometry cuvettes, and cuvettes may bedescribed as “receiving” a sample. Cytometry cuvettes or slides may havedifferent configurations.

In some embodiments, a cytometry cuvette may be configured to retain asample in a stationary position. These cuvettes may be referred toherein as “stationary sample cuvettes”. In such cuvettes, components ofa sample (e.g. cells, crystals, etc.) may, for example, settle to thefloor of the sample well of the cuvette, and may be sensed (e.g. imaged)while stationary on the floor of the sample well. In embodiments, two orthree-dimensional images of stationary sample may be obtained with animage sensor. Such images, for example, may be of cells or othercomponents of a sample. These images may provide a wealth of informationabout a selected component(s) of a sample. For example, in the case of acell, an image may provide, for example, information regarding cellsize, cell morphology, cell staining pattern, etc. Images of a sampleobtained with an image sensor may be analyzed with automated programs.

A schematic of an example cuvette for stationary sample analysis isprovided in FIG. 2. FIG. 2 shows a top-down view the exemplary cuvettefor stationary sample analysis. The exemplary cuvette 1101 contains foursample wells 1111, 1112, 1113, and 1114. A cuvette for stationary sampleanalysis may contain any number of sample wells, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, or more wells. A sample well may have any shapethat permits it to support a sample. For example, a sample well may becircular, elongated, square, etc. The sample wells may be fluidicallyisolated. Fluidically isolated wells may be advantageous, for example,to permit the loading of multiple different samples onto the samecuvette. Different samples may be, for example, samples from the samesubject that has been treated differently (e.g. stained with differentmarkers, dyes, etc.), or samples from different subjects. A sample wellmay have one or more ports 1121 for loading sample into the cuvette(each of the wells in FIG. 2 contains two ports; however, for clarity,only a single port is annotated in the Figure).

In some embodiments, a cytometry cuvette may be configured to supportthe flow of a sample from a first location to a second location withinthe cuvette. These cuvettes may be referred to herein as “flow samplecuvettes”. In flow sample cuvettes, a sample may, for example, move froma first location in the cuvette (e.g. the location where the sample isintroduced into the cuvette) to a second location in the cuvette (e.g. asample collection area). As a sample moves from a first location to asecond location in the cuvette, components of the sample (e.g. cells)may also move locations. In embodiments, a flow sample cuvette maycontain a port for introduction of sample into the cuvette, a samplecollection area, and a channel connecting the sample introduction portand the sample collection area. The channel may be enclosed. The channelmay have dimensions which are only a small amount larger than thediameter of cells of interest for analysis in the cuvette. For example,the channel may have a height, width, both height and width, or diameterof no greater than 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,or 100 microns. The channel of the cuvette may have any orientationrelative to the cuvette or the cytometry apparatus. For example,relative to a horizontal stage, the channel may be also be horizontal orit may be vertical or at an angle. Fluid may be propelled through thechannel by a variety of different forces, such as, for example,capillary action, gravity, positive pressure from a force behind theliquid, or negative pressure in front of the liquid. In embodiments,positive pressure may be exerted on a liquid in a channel by a pipettetip which loads sample into the channel through the sample introductionport and provides sufficient force on the sample to propel the samplethrough the channel. In embodiments, a sample collection area may havean air vent, to relieve pressure generated by the movement liquidtowards or into the sample collection area.

In embodiments, light from a light source may by directed at the channelof a flow sample cuvette. In embodiments, light from the objective maybe focused on or close to one or more particular location in thechannels of a flow sample cuvette (a “focal point” in the channel). At afocal point, light of a selected range of wavelengths from the lightsource may be focused. At a focal point in the channel, light from theobjective may be at a relatively high intensity, such that a greateramount of light may strike a cell at the focal point in the channel thanwould strike a cell in a stationary sample cuvette on the same cytometryapparatus. Accordingly, in some embodiments, when using the samecytometry apparatus, individual cells may be excited with greater lightintensity when they are in a flow sample cuvette than in a stationarysample cuvette. In some situations, it may be desirable to contact asample with a relatively high light intensity in order, for example, toincrease the intensity of scattered or emitted light from the sample.Increasing the intensity of light from a sample may, in turn, forexample, increase the speed or sensitivity at which a sample orcomponent therein may be detected.

In embodiments, an illumination train of a cytometry apparatus providedherein may be configured to focus light of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20 or more ranges of wavelengths at one or more differentlocations in a flow channel of a flow cytometry cuvette. For example, inorder to interrogate a sample and cells therein flowing through the flowchannel of a flow cytometry cuvette for fluorescence, it may bedesirable to focus light of a first range of wavelengths at a firstlocation in the channel, in order to excite a first type of fluorescentmarker which may be on cells which has a first peak (maximum) excitationwavelength and it may be desirable to focus light of a second range ofwavelengths at a second location in the channel, in order to separatelyexcite a separate type of fluorescent marker which may be on cells whichhas a second peak excitation wavelength. As is discussed further below,in embodiments, by spatially separating the locations at which a cellmay be exposed to focused light of different ranges of wavelengths,information from a cell may be efficiently obtained. A range ofwavelengths that may be focused may be, for example, a range of 200,100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5, or fewer nanometers.In embodiments, the range may be centered around the peak excitationwavelength of a particular fluorescent marker of interest. For example,the fluorophore fluorescein has a peak excitation wavelength of 494nanometers; a range of 20 nanometers around the 494 nanometer peak maybe selected as the range of wavelengths, such that light of 484-504nanometers is focused at a location in the channel of a flow samplecuvette. In another example, a range of 30 nanometers around the 494nanometer peak may be selected as the range of wavelengths, such thatlight of 479-509 nanometers is focused. Other ranges of wavelengths maybe selected based on other fluorescent markers, as appropriate. Inembodiments, a range of wavelengths may be, for example, selected basedon the color of the focused light, such as between 620-645 nanometers(red light), 520-550 nanometers (green light), 490-520 nanometers (cyanlight), or 460-490 (blue light).

In some embodiments, light emitted or scattered by a sample in a flowsample cuvette may be detected by a light detector of the cytometryapparatus, after the light has travelled through the objective of thecytometry apparatus and been directed to a detection train opticalpathway downstream of the objective which leads to one or more lightdetectors. For example, light detectors downstream of the objective maybe configured to detect emitted light from the sample of one or moreselected wavelengths. In embodiments, light detectors downstream of theobjective may be configured for detection of fluorescent light emissionfrom samples. Thus, in some embodiments, light detectors downstream ofthe objective may be used for detecting fluorescent molecules in asample in a flow sample cuvette. Typically, for fluorescence analysis ofsamples in a flow sample cuvette, light of one or more selectedwavelengths is directed to the sample from the objective in thecytometry apparatus. The light source for this light may be located inthe cytometry apparatus, and it may be filtered to contain only aselected range of wavelengths.

In embodiments, light emitted or scattered by a sample in a flow samplecuvette may be detected by a light detector of the cytometry apparatus.This light may provide, for example, information regarding the presenceof a marker on a cell, nucleic acid content of a cell, cell shape, etc.In embodiments, any information that can be obtained from a traditionalflow cytometer may be obtained with a cytometry apparatus and flowsample cuvette as provided herein.

In some embodiments, light emitted or scattered by a sample in a flowsample cuvette may be detected by a sample coplanar optical sensor. Aflow sample cuvette may contain one or more openings or optical pathwaysto permit light scattered or emitted from the sample in the channel ofthe flow sample cuvette to travel to a sample coplanar optical sensor. Aflow sample cuvette may additionally or alternatively contain one ormore openings or optical pathways to permit light from a sample coplanarlight source to reach the sample. In embodiments, an optical pathwayfrom the channel of the flow sample cuvette to a coplanar optical sensormay be at, for example, a 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, or 180 degree angle relative to an optical pathway to the channelfrom a coplanar light source. In some embodiments, a flow sample cuvettemay have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more openings or opticalpathways to permit light from a sample to travel to a sample coplanaroptical sensor. In a flow sample cuvette having two or more opticalpathways to a sample coplanar optical sensor, the optical pathways tothe sensors may be at different angles relative to an optical pathwayfrom a coplanar light source (e.g. if the flow sample cuvettes containstwo optical pathways to a sample coplanar optical sensor, the firstoptical pathway may be at a 15 degree angle relative to the opticalpathway from the coplanar light source, and the second optical pathwaymay be at a 45 degree angle relative to the optical pathway from thecoplanar light source). In some embodiments, as discussed above, throughthe use of fiber optics, different optical pathways in flow samplecuvette may be connected to the same optical sensor or light source. Insome embodiments, it may be advantageous to measure light scattering ofa sample with one or more sample coplanar optical sensors, rather thanwith one or more optical sensors which are downstream of the objective.For example, if a flow sample cuvette contains a narrow optical pathwayfrom the channel to an optical sensor, scattered light of a very limitedrange may be collected. Scatter information of a sample from a narrowoptical pathway may provide more information for sample analysis thatmay be provided from a wider scatter range. While the objective in acytometry apparatus may collect scattered light, typically, it may do sofrom a relatively large area, and thus, it may contain less information.

A schematic of an example cuvette for flow sample analysis is providedin FIG. 3. FIG. 3A shows a top-down view and FIG. 3B shows a side-viewof the exemplary cuvette for flow sample analysis. The exemplary cuvette1201 contains a sample introduction port 1211, flow channel 1212, andsample collection area 1213. Light from the objective may have one ormore focal point 1214 in the channel 1212. For example, in FIG. 3B,light of three selected ranges of wavelengths is focused onto threeseparate locations in the flow channel (collectively indicated by thethree dots 1214). As indicated in FIG. 3 by the direction of the arrow,sample flows from left to right in the example cuvette. FIG. 3B furthershows exemplary positions of a sample coplanar light source 1221 andoptical sensors 1222, 1223. The optical pathway of the first opticalsensor 1222 is at a 90 degree angle to the optical pathway of the lightsource 1221, while the optical pathway of the second optical sensor 1223is in-line (at an 180 degree angle) to the optical pathway of the lightsource 1221. Optical pathways to the sample coplanar light source andoptical sensors may intersect with at least one focal point 1214 in thechannel 1212.

In embodiments, when an illumination train of a cytometry apparatusprovided herein focuses light of two or more ranges of wavelengths attwo or more different locations in a flow channel of a flow cytometrycuvette, light emitted from objects present at the respective locationsmay be separately detected. For example, a cytometry apparatus providedherein may focus light of a first range of wavelengths on a firstlocation in a flow channel of a flow cytometry cuvette, and may focuslight of a second range of wavelengths on a second location in a flowchannel of a flow cytometry cuvette. As a sample containing cells isflowed through the flow channel, cells may pass through the firstlocation in the flow channel and then the second location in the flowchannel. The cells may be labeled with different fluorescent markers,which have different excitation peaks. The excitation peak of the firstfluorescent marker on the cells may overlap with the focused light ofthe first range of wavelengths, and the excitation peak of the secondfluorescent marker on the cells may overlap with the focused light ofthe second range of wavelengths. In such a circumstance, a cell labeledwith the first fluorescent marker will emit light at the firstfluorescent marker's emission spectrum when it is illuminated by thefocused light of the first range of wavelengths. Similarly, a celllabeled with the second fluorescent marker will emit light at the secondfluorescent marker's emission spectrum when it is illuminated by thefocused light of the second range of wavelengths. The light emitted bythe cell may be detected by one or more optical sensor. For example, inan embodiment, a cytometry apparatus and flow sample cuvette may beconfigured such that during the flow of a sample containing cellsthrough the flow channel of a flow sample cuvette, a single cell at atime passes through a region in the flow channel containing each of thedifferent locations in the flow channel where light of a range ofwavelengths. Such a region may be referred to as a “detection region”herein. For example, the flow channel of a flow sample cuvette on astage of a cytometry apparatus may have light of the wavelengths 390-420focused on a first location in the flow channel, it may have light ofthe wavelengths 460-490 focused on a second location in the flowchannel, and it may have light of the wavelengths 530-560 focused on athird location in the flow channel. These three locations and the spacebetween them may be collectively referred to as the “detection region”of the flow channel of the flow sample cuvette. Thus, in embodiments, acytometry apparatus and flow sample cuvette may be configured such thatduring the flow of a sample containing cells through the flow channel ofa flow sample cuvette, a single cell at a time passes through thedetection region. As the cell passes through the detection region, ifthe cell contains a fluorescent marker which is excited by a wavelengthof light focused on a respective location, the fluorescent marker on thecell will emit light which may be detected the cytometry apparatus. Forexample, the cell may be labeled with a first fluorescent marker whichhas a peak excitation wavelength of 400 nm and with a second fluorescentmarker which has a peak excitation wavelength of 545 nm. This cell willthus fluoresce when it passes through the first location and the thirdlocation (but not the second location) in the flow channel of theexample provided above. The emitted light from the cell can be detectedby a detection train as provided herein. For example, light from thedifferent locations in the detection region may be captured by theobjective and directed to a detection train containing one or more lightdetectors. In other embodiments, a cytometry apparatus and flow samplecuvette may be configured such that during the flow of a samplecontaining cells through the flow channel of a flow sample cuvette, onecell per location of focused light of a range of wavelengths in thedetection region flows through the detection region. For example, in theconfiguration described above (which has 3 locations in the flow channelwith a focused light of a range of wavelengths), the sample and systemmay be configured so that 3 cells flow through the detection region at atime. In such embodiments, at certain instances during the flow, onecell may be present at each of the different locations having focusedlight of a range of wavelengths. Such a set up may permit a more rapidanalysis of cells than a configuration in which only a single cell at atime is flowed through the detection region. In embodiments in whichmultiple cells flow through a detection region at the same time, lightemitted from cells may be detected through various detection trainconfigurations. In embodiments, the emitted light from multiple cells inthe detection region may be detected by light detectors in a detectiontrain comprising appropriate filters, lenses, mirrors, or other hardwarefor separating and detecting the relevant emitted light. In otherembodiments, the emitted light from multiple cells in the detectionregion may be detected by an image sensor in a detection traincomprising appropriate filters, lenses, mirrors, or other hardware forseparating and detecting the relevant emitted light. For example, anobjective may simultaneously capture light from cells in multipledifferent locations having focused light of a range of wavelengths in aflow channel. The light may be directed to an detection train whichincludes a prism or diffraction grating for separating light based onthe wavelength of the light. Such a detection train may permit a singleimage obtained by an image sensor to contain information regarding cellsat multiple different positions in a flow channel, each position being alocation having focused light of a range of wavelengths, and also inwhich the cells may emit light of different wavelengths. For example,FIG. 4 provides an exemplary schematic of an image that may be obtainedby an image sensor in a detection train as described immediately above.In the example for FIG. 4, an image is taken when one cell is at each ofthree different locations in the flow channel having focused light of arange of wavelengths. The focused range of wavelengths for the differentlocations may be the same or different. The light emitted from the cellpresent at each location is captured by the objective, and passedthrough a prism, diffraction grating, or other structure to separatelight based on wavelength. In FIG. 4, the image 1401 containsinformation from each of the three locations in the flow channel havingfocused light of a range of wavelengths. In the image 1401, along theX-axis positions 1411, 1412, and 1413 correspond to the three locationsin the flow channel having focused light of a range of wavelengths.Along the Y-axis, the numbers 400, 500, 600, and 700 indicatewavelengths of light. The parallel dotted lines for each of 1411, 1412,and 1413 are not in the actual image, but indicate a virtual line alongwhich the emitted light for each respective position may be located. Theblack dots 1421, 1422, and 1423 indicate the wavelength of light whichis sensed by the image sensor for each of the 1411, 1412, and 1413positions. Thus, in the exemplary image of FIG. 4, the cell present atthe first location in the flow channel emitted light having a peakintensity of approximately 650 nm, the cell present in the secondlocation in the flow channel emitted light having a peak intensity ofapproximately 410 nm, and the cell present in the third location in theflow channel emitted light having a peak intensity of approximately 550nm.

In embodiments in which the illumination train focuses light of two ormore ranges of wavelengths at two or more different locations in a flowchannel of a flow cytometry cuvette, the the illumination train mayremain in the same configuration regardless of whether the emitted lightfrom interrogated cells is to be detected by a light detector or animage sensor.

As an example of an analysis that may be performed with systems andmethod provided herein, a blood sample may be obtained from a subject.White blood cells may be isolated from the blood sample. The white bloodcells may be treated with three different antibodies, each antibodybeing directed to a different cell surface marker optionally present onthe surface of white blood cells (e.g. CD45, CD14, and CD3), eachantibody being labeled with a different fluorescent marker, and eachdifferent fluorescent marker having a different peak excitationwavelength. The sample may be flowed through a flow sample cuvette asprovided herein. As the sample flows through flow channel of thecuvette, cells (or other particles in the sample) may pass through threelocations in the flow channel which are exposed to focused light of arange of wavelengths. As the cells pass through each of the firstlocation, the second location, and the third location, the focused lightmay excite the fluorescent marker present on an antibody used to labelthe cell. If the fluorescent marker is excited, it may emit light at itsemission wavelength, and this light may be detected as describedelsewhere herein. The detected light may be analyzed to determineinformation regarding the cells flowed through the cuvette.

The cytometry apparatus may have a housing. The housing may contain oneor more of the components of the cytometry apparatus within the housing.In some embodiments, all of the components of the cytometry apparatusare within the housing. In some embodiments, the stage is on the surfaceof the housing, and cytometry cuvettes are supported outside thehousing. In some embodiments, cytometry cuvettes loaded onto a stageinside the housing.

In embodiments, methods and devices as provided herein may be used inmicro-gravity environments. For example, a flow sample cuvette asprovided herein may be used to perform sample assays in micro-gravityconditions. As described above, a flow sample cuvette contains a narrowchannel through which the sample moves and is exposed to light. Thischannel may be of a dimension such that cells or particles may onlytravel through the channel in single-file. Accordingly, a sample in aflow sample cuvette may be positioned in the cuvette in the same waywhether under standard gravity or micro-gravity environments.

Biological samples may be analyzed with systems and methods providedherein. The biological sample may be a bodily fluid, a secretion, or atissue sample. Examples of biological samples may include but are notlimited to, blood, serum, saliva, urine, gastric and digestive fluid,tears, stool, semen, vaginal fluid, interstitial fluids derived fromtumorous tissue, ocular fluids, sweat, mucus, earwax, oil, glandularsecretions, breath, spinal fluid, hair, fingernails, skin cells, plasma,nasal swab or nasopharyngeal wash, spinal fluid, cerebral spinal fluid,tissue, throat swab, biopsy, placental fluid, amniotic fluid, cordblood, emphatic fluids, cavity fluids, sputum, pus, microbiota,meconium, breast milk or other excretions. The sample may be providedfrom a human or animal. Samples may be from a plant, microorganism (e.g.virus, bacteria), or other biological material. Biological samples maycontain cells, crystals, particles, or other small objects. Cells in abiological sample may be separate from each other, or they may be linkedtogether. In embodiments, linked cells may be treated in order toseparate them into individual, free-moving cells. Cells may beprokaryotic or eukaryotic. In embodiments, a biological sample may be ablood sample, or a portion thereof. In embodiments, cells in abiological sample may be white blood cells. In embodiments, a bloodsample containing red blood cells and white blood cells may be treatedwith a reagent to lyse the red blood cells and stabilize white bloodcells. Such stabilized white blood cells may be analyzed with a systemor method provided herein.

In embodiments, cells may be prepared for cytometry by systems andmethods provided herein by any method known in the art. Cells may beoptionally fixed, stained, or otherwise labeled with a detectablemarker. Cells may be fixed with a variety of methods known in the art,including but not limited to heat, freeze, perfusion, immersion, andchemical fixation. Chemical fixation may be achieved by a wide varietyof agents, including but not limited to crosslinking agents (such asformaldehyde, glutaraldehyde, other aldehydes, and their derivatives),precipitating agents (such as ethanol and other alcohols), oxidizingagents (such as osmium tetroxide or potassium permanganate), potassiumdichromate, chromic acid, mercury-containing fixatives, acetic acid,acetone, picrates, and HOPE fixative. Cells may also be permeabilized,such as through the use of surfactants, as may be useful for subsequentinternal labeling or staining.

Cells may be stained with any optically detectable dye, stains, orcoloring agents, such as nucleic acid dyes (including intercalatordyes), lipophilic dyes, protein dyes, carbohydrate dyes, heavy metalstains. Such dyes and stains or staining processes include but are notlimited to Acid Fast Bacilli staining, Alcian Blue staining, AlcianBlue/PAS staining, Alizarin Red, alkaline phosphatase staining,aminostyryl dyes, ammonium molybdate, Azure A, Azure B, BielschowskyStaining, Bismark brown, cadmium iodide, carbocyanines, carbohydrazide,carboindocyanines, Carmine, Coomassie blue, Congo Red, crystal violet,DAPI, ethidium bromide, Diff-Quik staining, eosin, ferric chloride,fluorescent dyes, fuchsin, Giemsa stain, Golgi staining, Golgi-Coxstaining, Gomori's Trichrome staining, Gordon Sweet's staining, Gramstaining, Grocott Methenamine staining, haematoxylin, hexarnine, Hoechststains, Hyaluronidase Alcian Blue, indium trichloride,indocarbocyanines, indodicarbocyanines, iodine, Jenner's stain,lanthanum nitrate, lead acetate, lead citrate, lead(II) nitrate,Leishman stain, Luna staining, Luxol Fast Blue, Malachite green, MassonFontana staining, Masson Trichrome staining, methenamine, methyl green,methyline blue, microglia staining, Miller's Elastic staining, neutralred, Nile blue, Nile red, Nissl staining, Orange G, osmium tetroxide,Papanicolaou staining, PAS staining, PAS diastase staining, periodicacid, Perls Prussian Blue, phosphomolybdic acid, phosphotungstic acid,potassium ferricyanide, potassium ferrocyanide, Pouchet staining,propidium iodide (PI), Prussian Blue, Renal Alcian Blue/PAS staining,Renal Masson Trichrome staining, Renal PAS Methenamine staining,Rhodamine, Romanovsky stain, Ruthenium Red, Safranin O, silver nitrate,Silver staining, Sirius Red, sodium chloroaurate, Southgate'sMucicannine, Sudan staining, Sybr Green, Sybr Gold, SYTO dyes, SYPROstains, thallium nitrate, thiosemicarbazide, Toluidine Blue, uranylacetate, uranyl nitrate, van Gieson staining, vanadyl sulfate, von Kossastaining, WG staining, Wright-Giemsa stain, Wright's stain, X-Gal, andZiehl Neelsen staining. Cells may be treated with uncolored dyeprecursors that are converted to a detectable product after treatment,such as by enzymatic modification (such as by peroxidases orluciferases) or binding to an ion (such as Fe ions, Ca²⁺ or H⁺).

In embodiments, cells may be labeled with fluorescent markers. Usefulfluorescent markers include natural and artificial fluorescentmolecules, including fluorescent proteins, fluorophores, quantum dots,and others. Some examples of fluorescent markers that may be usedinclude but are not limited to: 1,5 IAEDANS; 1,8-ANS;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);fluorescein amidite (FAM); 5-Carboxynapthofluorescein;tetrachloro-6-carboxyfluorescein (TET); hexachloro-6-carboxyfluorescein(HEX); 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE); VIC®;NED™; tetramethylrhodamine (TMR); 5-Carboxytetramethylrhodamine(5-TAMRA); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX(carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-JOE; Light Cycler® red610; Light Cycler® red 640; Light Cycler® red 670; Light Cycler® red705; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; AutoFluorescent Proteins; Texas Red and related molecules;Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavinderivatives; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White);TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5);TRITC (TetramethylRodamine-IsoThioCyanate); True Blue; TruRed;Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; interchelatingdyes such as YOYO-3, Sybr Green, Thiazole orange; members of the AlexaFluor® dye series (from Molecular Probes/Invitrogen) such as Alexa Fluor350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610,633, 635, 647, 660, 680, 700, and 750; members of the Cy Dye fluorophoreseries (GE Healthcare), such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7;members of the Oyster® dye fluorophores (Denovo Biolabels) such asOyster-500, -550, -556, 645, 650, 656; members of the DY-Labels series(Dyomics), such as DY-415, -495, -505, -547, -548, -549, -550, -554,-555, -556, -560, -590, -610, -615, -630, -631, -632, -633, -634, -635,-636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680, -681,-682, -700, -701, -730, -731, -732, -734, -750, -751, -752, -776, -780,-781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL;members of the ATTO series of fluorescent labels (ATTO-TEC GmbH) such asATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 611X,620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740; members of theCAL Fluor® series or Quasar® series of dyes (Biosearch Technologies)such as CAL Fluor® Gold 540, CAL Fluor® Orange 560, Quasar® 570, CALFluor® Red 590, CAL Fluor® Red 610, CAL Fluor® Red 635, Quasar® 570, andQuasar® 670.

Fluorescent markers may be coupled to a targeting moiety to allowspecific binding or localization, for example, to a specific populationof cells, of which there are many known in the art. Nonlimiting examplesinclude antibodies, antibody fragments, antibody derivatives, aptamers,oligopeptides such as the nuclear localization sequence (NLS), smallmolecules that serve as specific ligands for receptors including manyhormones and drugs, nucleic acid sequences (such as for FISH), nucleicacid binding proteins (including repressors and transcription factors),cytokines, ligands specific for cellular membranes, enzymes, moleculesthat specifically bind to enzymes (such as inhibitors), lipids, fattyacids, and members of specific binding interactions such asbiotin/iminobiotin and avidin/streptavidin.

Targets for specific labeling in or on a cell may be natural orartificial and may encompass proteins, nucleic acids, lipids,carbohydrates, small molecules, and any combinations thereof. Theseinclude intracellular and cell surface markers. Intracellular markersinclude any molecule, complex, or other structure within the cell. A fewnonlimiting examples include genes, centromeres, telomeres, nuclear porecomplexes, ribosomes, proteasomes, an internal lipid membrane,metabolites such as ATP, NADPH, and their derivatives, enzymes or enzymecomplexes, protein chaperones, post-translational modifications such asphosphorylation or ubiquitinylation, microtubules, actin filaments, andmany others. Cell surface markers include but are not limited toproteins such as CD4, CD8, CD45, CD2, CRTH2, CD19, CD3, CD14, CD36,CD56, CD5, CD7, CD9, CD10, CD11b, CD11c, CD13, CD15, CD16, CD20, CD21,CD22, CD23, CD24, CD25, CD33, CD34, CD37, CD38, CD41, CD42, CD57, CD122,CD52, CD60, CD61, CD71, CD79a, CD95, CD103, CD117, CD154, GPA, HLA, KOR,FMC7. In some embodiments, the targets may be specific regions within acell, such as targeting to the interior of specific organelles ormembrane-bound vesicles. In some embodiments, the target may be theresult of genetic or other manipulation, such as cloning Lac bindingsites into a genetic sequence for targeted binding by a labeled Lacprotein.

Cells may be labeled through various means, including but not limited tosurface labeling, permeabilization of the cell membrane and/or cellwall, active transport or other cellular processes, diffusion throughthe membrane, carrier particles such as lipid vesicles or otherhydrophobic molecules, and production by the cell (such as forrecombinantly fluorescent proteins).

In some embodiments, samples containing mixed populations of cells maybe treated before optical detection to enrich for detection of targetpopulation(s) of cells. Some example methods for enrichment include butare not limited to centrifugation, sorting (with or without labeling),selective killing of non-target cells such as by lysis, and selectivelabeling to improve detection of target cells.

In embodiments, cells for use with methods or devices provided hereinmay be prepared or analyzed (e.g. isolated, washed, stained, imaged,etc.) as described in any of U.S. Provisional Patent App. Nos.61/675,811, 61/676,178, 61/766,116, 61/802,194, each of which isincorporated by reference in their entirety. In some embodiments,devices or methods disclosed herein may be used with any of systems,methods, or devices as disclosed in, for example, U.S. Pat. No.8,380,541; U.S. Pat. App. Ser. No. 61/675,811, filed Jul. 25, 2012; U.S.Patent Application 61/837,168, filed Jun. 19, 2013, entitled “METHODSAND DEVICES FOR SMALL VOLUME LIQUID CONTAINMENT”; U.S. PatentApplication 61/837,151, filed Jun. 19, 2013, entitled “DEVICES, SYSTEMS,AND METHODS FOR CELL ANALYSIS IN MICROGRAVITY”; U.S. Pat. App. Ser. No.61/676,178, filed Jul. 26, 2012; U.S. Pat. App. Ser. No. 61/766,116,filed Feb. 18, 2013; U.S. Pat. App. Ser. No. 61/802,194, filed Mar. 15,2013; U.S. patent application Ser. No. 13/769,798, filed Feb. 18, 2013;U.S. patent application Ser. No. 13/769,779, filed Feb. 18, 2013; U.S.patent application Ser. No. 13/244,947 filed Sep. 26, 2011;PCT/US2012/57155, filed Sep. 25, 2012; U.S. application Ser. No.13/244,946, filed Sep. 26, 2011; U.S. patent application Ser. No.13/244,949, filed Sep. 26, 2011; and U.S. Application Ser. No.61/673,245, filed Sep. 26, 2011, the disclosures of which patents andpatent applications are all hereby incorporated by reference in theirentireties.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. Any feature, whetherpreferred or not, may be combined with any other feature, whetherpreferred or not. It should also be understood that while the inventionprovided herein has been described herein using a limited number ofterms and phrases for purposes of expediency, the invention could alsobe described using other terms and phrases not provided herein whichalso accurately describe the invention. The appended claims are not tobe interpreted as including means-plus-function limitations, unless sucha limitation is explicitly recited in a given claim using the phrase“means for.” It should be understood that as used in the descriptionherein and throughout the claims that follow, the meaning of “a,” “an,”and “the” includes plural reference unless the context clearly dictatesotherwise. For example, a reference to “an assay” may refer to a singleassay or multiple assays. Also, as used in the description herein andthroughout the claims that follow, the meaning of “in” includes “in” and“on” unless the context clearly dictates otherwise. As used in thedescription herein and through the claims that follow, a first objectdescribed as containing “at least a portion” of a second object maycontain the full amount of/the complete second object. As used in thedescription herein and throughout the claims that follow, the terms“comprise”, “include”, and “contain” and related tenses are inclusiveand open-ended, and do not exclude additional, unrecited elements ormethod steps. Finally, as used in the description herein and throughoutthe claims that follow, the meaning of “or” includes both theconjunctive and disjunctive unless the context expressly dictatesotherwise. Thus, the term “or” includes “and/or” unless the contextexpressly dictates otherwise.

This document contains material subject to copyright protection. Thecopyright owner (Applicant herein) has no objection to facsimilereproduction by anyone of the patent documents or the patent disclosure,as they appear in the US Patent and Trademark Office patent file orrecords, but otherwise reserves all copyright rights whatsoever. Thefollowing notice shall apply: Copyright 2013-14, Theranos, Inc.

We claim:
 1. A system for processing a biological sample, the systemcomprising: a flow sample cuvette; and a cytometry apparatus comprisingan illumination train, a detector, another detector, and a stage forreceiving the flow sample cuvette, wherein the flow sample cuvette isconfigured to receive and support a biological sample, wherein the flowsample cuvette contains a flow channel wherein the illumination traincomprises a light source, an objective of the stage, and a samplecoplanar light source disposed to be substantially coplanar with theflow channel, wherein light from said sample coplanar light source isnot in optical communication with the objective, wherein the objectiveis optically coupled to said stage such that light from the biologicalsample enters the objective; wherein during the flowing of samplethrough the flow channel, light of a first range of wavelengths from thelight source is focused on a first location in the flow channel andlight of a second range of wavelengths from the light source is focusedon a second location in the flow channel; and wherein the detector ispositioned to detect light emitted from a cell from the biologicalsample in the first location in the flow channel and another detector ispositioned to detect light emitted from another cell from the biologicalsample in the second location in the flow channel.
 2. The system ofclaim 1, wherein the cell is a white blood cell.
 3. The system of claim1, wherein the first location and second location each have a diameterbetween 10 and 200 microns.
 4. The system of claim 1, wherein the firstlocation and the second location each have a center, and wherein thecenter of the first location is separated from the center of the secondlocation by a distance between 10 and 200 microns.
 5. The system ofclaim 1, wherein the first location and the second location each have acenter, and wherein the center of the first location is separated fromthe center of the second location by a distance of at least an averagediameter size of cells in the biological sample.
 6. The system of claim1, wherein the detector comprises at least 3 image sensors.
 7. Thesystem of claim 6, wherein a first of the 3 image sensors is configuredto receive light of a first range of wavelengths, a second of the 3image sensors is configured to receive light of a second range ofwavelengths, and a third of the 3 image sensors is configured to receivelight of a third range of wavelengths.
 8. The system of claim 6, whereinemission filters are positioned before each of the at least 3 imagesensors.
 9. The system of claim 1, wherein for first illumination outputthe illumination train is further configured to focus light of a thirdrange of wavelengths from the light source on a third location of theflow sample cuvette.
 10. The system of claim 1, wherein the firstlocation in the flow channel and the second location in the flow channelare separated from each other by at least 10 and no more than 100microns.
 11. A system for processing a biological sample, the systemcomprising: a flow sample cuvette; a cytometry apparatus comprising anillumination train, a detector, another detector, and a stage forreceiving the flow sample cuvette, wherein the flow sample cuvette isconfigured to receive and support a biological sample, wherein the flowsample cuvette contains a flow channel wherein the illumination traincomprises a light source, an objective of the stage, and a samplecoplanar light source disposed to be substantially coplanar with theflow channel, wherein light from said sample coplanar light source isnot in optical communication with the objective, wherein the objectiveis optically coupled to said stage such that light from the biologicalsample enters the objective; and a stationary sample cuvette to thestage of the cytometry apparatus, wherein the stationary sample cuvetteis configured to receive and support a biological sample, wherein thestationary sample cuvette comprises a well for holding the biologicalsample in a stationary position, wherein the well comprises side wallsand a bottom surface, and wherein the bottom surface of the wellcomprises a sample plane on which the biological sample and objectstherein may settle; wherein during the flowing of sample through theflow channel, light of a first range of wavelengths from the lightsource is focused on a first location in the flow channel and light of asecond range of wavelengths from the light source is focused on a secondlocation in the flow channel; and wherein the detector is positioned todetect light emitted from a cell from the biological sample in the firstlocation in the flow channel and another detector is positioned todetect light emitted from another cell from the biological sample in thesecond location in the flow channel.
 12. A system for processing abiological sample, the system comprising: a flow sample cuvette; and acytometry apparatus comprising an illumination train, a detector,another detector, and a stage for receiving the flow sample cuvette,wherein the flow sample cuvette is configured to receive and support abiological sample, wherein the flow sample cuvette contains a flowchannel wherein the illumination train comprises a light source, anobjective of the stage, and a sample coplanar light source disposed tobe substantially coplanar with the flow channel, wherein light from saidsample coplanar light source is not in optical communication with theobjective, wherein the objective is optically coupled to said stage suchthat light from the biological sample enters the objective; whereinduring the flowing of sample through the flow channel, light of a firstrange of wavelengths from the light source is focused by the objectiveon a first location in the flow channel and light of a second range ofwavelengths from the light source is focused by the objective on asecond location in the flow channel; wherein the detector is positionedto detect light emitted from a cell from the biological sample in thefirst location in the flow channel and said light passing through theobjective before reaching the detector; and wherein another detector ispositioned to detect other light emitted from another cell from thebiological sample in the second location in the flow channel and saidother light passing through the objective before reaching said anotherdetector.